By now, several methods have been described for assaying a nucleotide sequence and identifying individual substitutions of bases by hybridization techniques (Cotton, R. G. H. // Biochem. J, 1989, V. 263, pp. 1-10).
The most widespread techniques are those in which a test DNA fragment is attached to a membrane and hybridized thereon with labelled oligonucleotides (Wallace, P. B., Shaffer, J., Murphy, R. F., Bonner, J., Hirose, T., Itakura, K. //Nucleic Acid Res., 1979, V. 6 pp. 3543-3557).
Known in the art are a method and a device for determining a DNA nucleotide sequence (E. Southern et al., PCT/GB 89/00460, 1989), which method comprises synthesizing oligonucleotides on a glass support, effecting hybridization with radioactively- or fluorescently-labelled test DNA, washing in the duplex dissociation conditions, detecting the presence of individual substitutions in the test sequence by analyzing the autoradiographic patterns or the intensity of fluorescence at individual dots, and reconstructing the DNA nucleotide sequence on the basis of data analysis. A device for carrying out said method comprises a supporting film or glass plate and a matrix covalently attached to the surface thereof, the matrix comprising the whole set or a selected part of oligonucleotides of desired length, the latter oligonucleotides being capable of taking part in the hybridization reactions. The surface of the support to which the oligonucleotides are attached is made of glass.
The above method and device, however, have low sensitivity. The value of the signal from the labelled DNA (laid out in the near-surface layer of the support) obtained from each individual matrix element is limited by the substrate surface capacity (as regards the covalently attached oligonucleotides) and it cannot be raised without increasing either the area of the matrix element or the sensitivity of the labelling marker. These limitations reduce the resolving power of the method and device, make it difficult to miniaturize the matrix, increase the requirements imposed on the sensitivity and resolving power of the detector, and raise the consumption of the reagents. The method is rather complicated because, even in assaying a test DNA fragment, it requires a series of successive hybridizations to be performed, with additional rounds of oligonucleotide matrix synthesis to be performed at one-letter step, for each dot where hybridization has not yielded unambiguous information on the sequence and, therefore each time new optimal hybridization conditions (temperature, reagent concentrations, etc.) have to be chosen, which involves further experimentation and considerable expenditure of time and reagents.